Clinical and microbiological characteristics of persistent Staphylococcus aureus bacteremia, risk factors for mortality, and the role of CD4+ T cells

This study evaluated the determinants of mortality and the T cell immune response in patients with persistent Staphylococcus aureus bacteremia (SAB). This was a prospective cohort study and patients with confirmed SAB were enrolled from 2008 to 2020. We compared clinical, microbiological, and genotypic features between surviving and deceased patients with persistent SAB. The concentrations of cytokines and the proportions of IFN-γ secreting CD4+ T cells were measured serially during the bacteremia period. Of the 1760 patients, 242 had persistent bacteremia (PB), and 49 PB patients died within 30 days. In the multivariate analysis, the APACHE II score and female sex were independently associated with 30 days mortality. The level of IL-10 was significantly increased in the plasma of patients with a high Pitt bacteremia score and those who died within 12 weeks from the index day. The proportion of IFN-γ-secreting CD4+ T cells were the highest just before the positive-to-negative conversion of blood cultures in patients with a low Pitt bacteremia score and those who survived for 12 weeks. The level of IL-10 is correlated with clinical outcomes in PB patients. IFN-γ secreting CD4+ T cells might play a pivotal role in SAB PB.

Staphylococcus aureus is a leading cause of serious bacterial infections 1,2 .S. aureus bacteremia (SAB) may persist in some patients despite appropriate antibiotic therapy.The use of intravascular catheters or prosthetic devices, metastatic infection, methicillin resistance, vancomycin minimal inhibitory concentration (MIC), and accessory gene regulator (agr) dysfunction have been suggested as risk factors for persistent bacteremia (PB) [3][4][5][6] .Several reports have identified clinical outcomes associated with PB compared with resolving bacteremia (RB).In patients with SAB, PB is typically associated with complications of bacteremia, longer hospitalization, and increased mortality than RB 4,[6][7][8][9] .Factors that predict mortality must be elucidated to determine the optimal strategy for treating S. aureus PB.
The outcome of S. aureus infection could be influenced by the host immune response to S. aureus.The cytokine response to S. aureus infection appears to be related to clinical outcomes.IL-6 is an early inflammatory marker of complicated SAB, and IL-10 is associated with mortality and PB [10][11][12][13] .Also, previous studies have attempted to establish the contribution of T cell immunity during S. aureus infection, along with efforts to develop a vaccine against S. aureus infection 14,15 .CD4 + T helper (Th) cell immunity might be important for fighting S. aureus infections in humans 16 .In particular, Th1 cells produce IFN-γ, which promotes bacterial elimination inside macrophage phagosomes and plays an important role in immunological and inflammatory processes during S. aureus infection 14,[16][17][18] .
Although there have been several reports evaluating the mortality of SAB and risk factors for PB, the factors affecting the mortality of patients with PB remain unclear.In addition, most studies of T cell immunity during

Study definitions
PB was defined as bacteremia for ≥ 7 days while receiving appropriate antibiotics therapy.The place of acquisition was categorized as community-acquired, healthcare-associated, or hospital-acquired infection 23 .Infective endocarditis was defined according to the modified Duke criteria 24 .Two sets of cultures were obtained to diagnose incident bloodstream infections.The index day was defined as the day on which the first positive blood culture was obtained.Empirical antibiotic treatment was considered appropriate if at least one antibiotic effective against the SAB isolate was started within 24 h after the index blood culture.Prosthetic devices included orthopedic devices, cardiovascular electronic devices, prosthetic valves, and vascular grafts.Septic shock was defined as sepsis with persisting hypotension that required vasopressors to maintain the patient's blood pressure despite adequate fluid resuscitation 25 .The concentrations of cytokines and the proportion of IFN-γ secreting CD4 + T cells were compared according to the patient's bacteremia severity scores and 12-week mortality.The patients were divided into groups: Group 1 had Pitt bacteremia score < 4, and Group 2 had Pitt bacteremia score ≥ 4. Group 3 was patients who survived for 12 weeks from the index day, and Group 4 was patients who died within 12 weeks.

Microbiological analysis
All S. aureus isolates were identified by standard methods.The first blood isolate obtained from the patient was used for the microbiological and molecular assessments.The minimum inhibitory concentration (MIC) of vancomycin was determined using a broth microdilution method (BMD) according to the standard protocol.Methicillin resistance was confirmed by detecting the mecA gene via polymerase chain reaction (PCR).
The staphylococcal cassette chromosome mec (SCCmec) type and agr genotype were identified as previously described 26,27 .The agr function was determined by the δ-hemolysin activity as described elsewhere 28 .Multi-locus sequence typing (MLST) was performed for all strains as described previously 29 .MLST allele names and STs were derived from the MLST database (http:// www.mlst.net).Clonal complexes (CCs) were assigned to groups of isolates that shared six of seven alleles via eBURST (http:// eburst.mlst.net).

Cytokine assays
The concentrations of IL-6, IL-10, IL-17A, IL-12p70, IL-13, IL-4, TNF-α, and IFN-γ in plasma were measured with a ProcartapPlex Multiplex Immunoassay (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.If the level for a particular cytokine was below the detectable limit, the concentration was recorded as 0.

Statistical analysis
We compared surviving and deceased patients with PB.Categorical variables were compared using the Chisquare test or Fisher's exact test, as appropriate.Continuous variables were compared using Student's t test and the Mann-Whitney U-test.Logistic regression was used to assess risk factors associated with PB mortality.Risk factors for 30 days mortality from PB were assessed using univariate analysis, and statistically significant variables were included in the multivariate analysis.Two-way ANOVA with Bonferroni post-tests was used to analyze the cytokine kinetics.A two-tailed p value < 0.05 was considered significant.All statistical analyses were performed using IBM SPSS Statistics for Windows, version 25 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 9.4.1 (GraphPad Software, Inc., San Diego, CA, USA; www.graph pad.com).

Ethics declarations
The study was approved by the Institutional Review Board (IRB) of Asan Medical Center (protocol No. 2013-1002).Informed consent was obtained from all individual participants included in the research.The study was performed in accordance with the Helsinki Declaration.

Clinical characteristics and outcomes of patients with PB
From July 2008 to December 2020, 1760 episodes of SAB were reported.Among these 1760 episodes, 242 (

Risk factors for 30 days mortality of patients with PB
A multivariate logistic regression analysis was performed to identify independent risk factors associated with 30 days mortality from PB (Table 2).The multivariate analysis indicated that the APACHE II score (adjusted odds ratio [aOR], 1.10; 95% confidence interval [CI] 1.03-1.17)and female sex (aOR 3.84; 95% CI 1.40-10.54)were independent risk factors for 30 days mortality from PB.

Risk factors for 30 days mortality from MRSA PB
A multivariate logistic regression analysis was employed to identify independent risk factors associated with 30 days mortality from MRSA PB (Supplementary Table S3).The multivariate analysis indicated that the APACHE II score (aOR 1.07; 95% CI 1.01-1.13),liver cirrhosis (aOR 3.77; 95% CI 1.43-9.95),and a vancomycin MIC of ≥ 1.5 mg/L (aOR 3.17; 95% CI 1.39-7.25)were independent risk factors for 30 days mortality in MRSA PB.

Kinetics of IL-6 and IL-10 in patients with PB
Among 242 patients with PB, 22 patients had blood samples collected periodically.Clinical backgrounds and outcomes of the 22 patients are listed in Supplementary Table S4.Their IL-6, IL-10, IL-17A, IL-12p70, IL-13, IL-4, and TNF-α plasma concentrations were analyzed.One patient died within 30 days, and three patients died within 12 weeks of the index day.IL-12p70, IL-13, and IL-4 levels were undetectable in most patients.IL-17A and TNF-α levels were measured in only eight and two patients, respectively.Therefore, IL-17A, IL-12p70, IL-13, IL-4, and TNF-α were excluded from the analysis.The IL-10 level could not be detected in three patients, and thus only 19 patient samples were available for the analysis of IL-10.Changes in the plasma levels of IL-10 and  We compared groups according to the Pitt bacteremia score and 12-week mortality (Figs. 2 and 3).IL-10 concentrations were higher in patients with high bacteremia scores than those with low bacteremia scores 4-9 days prior to the last positive culture day (Fig. 2, p < 0.05).The concentrations of IL-10 in the 4-9 days before the last positive culture day were higher in patients who died within 12 weeks from the index day than those who  www.nature.com/scientificreports/survived (Fig. 3, p < 0.05).IL-6 concentrations were generally higher in patients with higher Pitt bacteremia scores and those who died within 12 weeks, but the differences between groups did not reach statistical significance.

Kinetics of IFN-γ and CD4 + T cells secreting IFN-γ in patients with PB
IFN-γ was undetectable in the plasma of 22 patients from whom blood samples were collected periodically (Supplementary Table S4).We collected PBMCs from these 22 patients to analyze IFN-γ further.After stimulation of the PBMCs with heat-killed S. aureus, the IFN-γ in the cell culture medium was detected for PBMCs isolated from 19 patients.IFN-γ concentrations increased until just before the blood cultures underwent a negative conversion and decreased after the last positive blood culture day for 15 patients with low bacteremia scores (Fig. 4a).IFN-γ concentrations were very low for all time points for four patients with high bacteremia scores.
To determine whether CD4 + T cells produce IFN-γ, intracellular staining was performed, and IFN-γ secreting cells among the CD4 + T cells of 16 patients could be analyzed by using flow cytometry.The proportion of IFN-γ secreting CD4 + T cells tended to be correlated with the IFN-γ concentration and was significantly high 1-3 days before the last positive culture day for 15 patients with low bacteremia scores (Fig. 4b, group 1).IFN-γ secreting CD4 + T cells were identified for only one patient with a high bacteremia score (Fig. 4b, group 2).When comparing groups of patients according to the 12-week mortality, the concentration of IFN-γ increased until the blood culture became negative and decreased after the last positive culture day in both groups (Fig. 4c).In addition, the proportion of IFN-γ secreting cells among the CD4 + T cells was the highest 1-3 days before the blood culture became negative for 13 patients who survived for 12 weeks from the index day (Fig. 4d, group 3). Figure 5 shows the proportion of IFN-γ secreting cells among the CD4 + T cells analyzed using flow cytometry 1-3 days before the last positive culture day.

Discussion
In this study, we evaluated the risk factors associated with mortality in patients with SAB PB and confirmed a T cell immune response during S. aureus infection.In particular, our study is meaningful in that it sequentially observed changes in cytokines and T cell responses over time in human SAB.The APACHE II score and female sex were independent risk factors of 30 days mortality.The subgroup analysis according to MRSA isolates showed that the APACHE II score, liver cirrhosis, and a vancomycin MIC of ≥ 1.5 mg/L were independent risk factors for 30 days mortality.The IL-10 concentration was high in the early phase of bacteremia in patients with high Pitt bacteremia scores and those who died within 12 weeks from the index day.The proportion of IFN-γ-secreting CD4 + T cells was highest just before the blood cultures turned negative for patients with low Pitt bacteremia scores and those who survived for 12 weeks.
Previously, there have been several studies showing the risk factors of SAB PB.Patients with liver cirrhosis have impaired immunity and increased susceptibility to S. aureus infections.S. aureus is an important pathogen in liver cirrhosis patients 31,32 .Liver cirrhosis is a risk factor for PB compared with RB; moreover, liver cirrhosis patients with SAB had a higher mortality rate than non-liver cirrhosis patients in previous studies 8,33 .A high vancomycin MIC is also associated with worse clinical outcomes and treatment failure but there has been a controversy about whether a high vancomycin MIC is associated with persistent bacteremia 4,8,34,35 .Sheng-Hsiang Lin et al. 36 showed that a decreased susceptibility to vancomycin was associated with mortality in MRSA PB.In this study, we evaluated the risk factors associated with 30 days mortality in patients with SAB PB.Liver cirrhosis was identified as a risk factor for 30 days mortality in patients with MRSA PB.In addition, the surviving patients were more likely to have a lower vancomycin MIC, and a vancomycin MIC of ≥ 1.5 mg/L was an independent risk factor for 30 days mortality from MRSA PB.Decreased susceptibility to vancomycin appears to affect the mortality of patients with MRSA PB treated with glycopeptides including vancomycin.
The agr locus of S. aureus globally controls the coordinated production of virulence factors 27 .Mutations in the agr locus cause agr dysfunction, alter the expression of autolysins and hemolysins, and have a global effect on bacterial pathogenicity 37,38 .agr dysfunction has been associated with PB, vancomycin bactericidal activity, and the mortality of SAB patients 6,28,39 .Loss of agr function appears to decrease the susceptibility to glycopeptides 40 .www.nature.com/scientificreports/Moise-broder et al. 41 reported that agr group II was a predictor of vancomycin treatment failure of MRSA bacteremia and Park et al. 42 showed that agr group II was more pronounced in MRSA isolates with a vancomycin MIC of 2 mg/L.In the present study, agr dysfunction was noted in 64.9% of MRSA PB cases and there was no difference between the two groups.agr group II was the most common genotype of MRSA PB, and although statistically insignificant, agr group II was more frequent in patients who died within 30 days from the index day than in surviving patients.Additional studies are necessary to investigate further the association of mortality in PB patients with the agr group and agr dysfunction.Pro-inflammatory cytokines such as IL-6, tissue necrosis factor (TNF), and IL-17A are necessary for initiating an effective inflammatory process against infection and they are associated with multiple-system organ failure and mortality 43 .IL-10 is an anti-inflammatory cytokine that regulates the immune response to pathogens 44 .Previous studies have tried to show a correlation between cytokines and SAB.McNicholas et al. 12 reported that IL-6, a pro-inflammatory cytokine, might be an early inflammatory marker of complicated SAB.IL-10 was found to be associated with mortality and persistent bacteremia 10,11,13 .It is significant that, unlike previous studies, we measured IL-6 and IL-10 concentrations serially throughout the bacteremia period.Although there was no significant difference, the plasma concentration of IL-6 generally tended to be high in patients with high Pitt bacteremia scores and those who died within 12 weeks, before the blood culture became negative.The plasma concentration of IL-10 4-8 days before the last positive blood culture day was significantly higher in patients with high bacteremia scores and patients who died within 12 weeks from the index day.The IL-10 concentration in the early stage of bacteremia can be a predictive factor of poor clinical outcomes of persistent bacteremia.
There have been several lines of evidence showing that T cell immunity plays an important role in S. aureus infection.T lymphocyte-deficient mice were more susceptible to infection with S. aureus, and T cell-derived IFN-γ might be a pivotal regulator of neutrophil recruitment during S. aureus infection in a mouse model 45,46 .Human immunodeficiency virus infected patients with impaired Th1 cell immunity and low CD4 + counts are highly susceptible to S. aureus and have more invasive infections and a higher recurrence rate than patients with www.nature.com/scientificreports/intact T cell immunity [47][48][49][50][51] .The first study showing the activation of a T cell immune response in human S. aureus infection was published in 2015.Brown et al. 30 showed that human S. aureus blood stream infection induces S. aureus antigen-specific IFN-γ-producing CD4 + (Th1) cells.
In our study, we attempted to ascertain the T cell immune response within a real world clinical setting in patients with SAB PB.After stimulation of PBMCs from PB patients with heat-killed S. aureus, we were able to analyze the concentrations of secreted IFN-γ in the cell culture medium.The concentration of IFN-γ was highest 1-3 days before the blood culture became negative, and it decreased after the last positive blood culture day in patients with low disease severity compared to those with severe disease.In addition, the proportion of IFNγ-producing CD4 + Th1 cells showed similar kinetics to the IFN-γ concentration during infection.Translating these findings, we found that CD4 + T cells are an important source of IFN-γ production during bacteremia and they play an important role in bacterial clearance and contribute to disease severity in SAB PB.While previous studies have shown a potential for T cell immunity to play a protective role against SAB infection, our study further clarifies the role of T cell immunity in S. aureus PB by showing the kinetics of CD4 + T cells during S. aureus bacteremia.
Our study has several limitations.First, the patients with a long duration of bacteremia were likely to be managed in great detail by infectious disease specialists, which included receiving additive diagnostic tests and antibiotic treatment.These actions might have resulted in a bias against the outcome of PB.Second, because the definition of PB is seven days, the possibility of an immortal time bias cannot be overlooked.Further analysis is needed to reduce immortal time bias and determine the presence of differences in outcomes depending on the duration of bacteremia.Third, only a small number of PB patients had serial blood samples collected.

Figure 1 .
Figure 1.Cytokine kinetics during the bacteremia period.Plasma concentrations of IL-10 and IL-6 were analyzed in serial blood samples from 19 and 22 patients, respectively.(a) and (b) are the mean concentrations of IL-10 and IL-6 during the bacteremia period.Day 0 is the last positive culture day.

Figure 2 .
Figure 2. Comparison of cytokine concentrations in groups 1 and 2 in serial blood samples collected during the study period.(A) IL-10 concentration in plasma.Group 1 comprises 15 patients, and group 2 comprises four patients.(B) IL-6 concentration in plasma.Group 1 comprises 18 patients, and group 2 comprises 4 patients.Day 0 was the last day of positive culture.*p < 0.05.Group 1 comprises patients with a Pitt bacteremia score of < 4. Group 2 comprises patients with a Pitt bacteremia score of ≥ 4.

Figure 3 .
Figure 3.Comparison of cytokine concentrations in group 3 and 4 in serial blood samples collected during the study period.(A) IL-10 concentration in plasma.Group 3 comprises 15 patients, and group 4 comprises four patients.(B) IL-6 concentration in plasma.Group 3 comprises 18 patients, and group 4 comprises 4 patients.Day 0 was the last day of positive culture.*p < 0.05.Group 3 comprises patients who survived for 12 weeks from the index day.Group 4 comprises patients who died within 12 weeks from the index day.

Figure 4 .
Figure 4. Comparison between groups of IFNγ concentrations and the proportion of IFNγ-secreting cells among the CD4 + T cells in patients with serial blood samples collected during the study period.PBMCs were collected from 22 patients and stimulated with heat-killed S. aurerus, and the concentration of IFNγ was measurable in 19 patients.In addition, the proportion of IFNγ-secreting cells among the CD4 + T cells was assessed using flow cytometry for 16 patients.(A) IFNγ concentration in Group 1 and Group 2. (B) The proportion of IFNγ-secreting cells among CD4 + T cells in Group 1 and Group 2. (C) IFNγ concentration in Group 3 and Group 4. (D) the proportion of IFNγ-secreting cells among CD4 + T cells in Group 3 and Group 4. Day 0 was the last day of positive culture.*p < 0.05.Group 1 comprises patients with Pitt bacteremia score < 4. Group 2 comprises patients with Pitt bacteremia ≥ 4. Group 3 comprises patients who survived for 12 weeks from the index day.Group 4 comprises patients who died within 12 weeks from the index day.

Figure 5 .
Figure 5. Proportion of IFNγ-secreting cells among CD4 + T cells analyzed using flow cytometry 1-3 days before the last positive culture day.Group 1 comprises 15 patients, and group 2 comprises one patient.Group 3 comprises 13 patients, and group 4 comprises 3 patients.Group 1 comprises patients with Pitt bacteremia score < 4. Group 2 comprises patients with Pitt bacteremia ≥ 4. Group 3 comprises patients who survived for 12 weeks from the index day.Group 4 comprises patients who died within 12 weeks from the index day.

Table 1 .
Demographic and clinical characteristics of patients with persistent S. aureus bacteremia.IQR, interquartile range; APACHE II, acute physiology and chronic health evaluation II; CVC, central venous catheter.a Includes orthopedic devices (12 patients), cardiovascular implantable electronic devices (3 patients), prosthetic valves (12 patients), and vascular grafts (27 patients).b Echocardiography was performed in 94.6% (229/242) of patients with persistent bacteremia.c Eradicable focus refers to a removable source of infection, such as a vascular catheter, vascular graft, prosthetic device, or abscess.

Table 2 .
Multivariate analysis of the risk factors for 30 days mortality in patients with persistent S. aureus bacteremia.a This model fits the data well in terms of discrimination (C-statistic 0.769) and calibration (Hosmer-Lemeshow goodness-of-fit statistic 9.492; p = 0.303).